Bioreactor with scaffolds

ABSTRACT

A bioreactor for culturing of cells is described. Screens suitable as a cell growth scaffold may comprise crossed fibers. Screens may be contained loosely in a screen holder, which in turn may be contained inside a manifold assembly. A lower manifold, screen holder and upper manifold may have identical or similar interior open cross-sections. Flow of liquid medium can occur upwardly through the array of screens, then flowing over a weir in the presence of an air pocket, and into a moat and a pump. The screen holder may have slots whose exterior-facing ligaments are rounded, and may have grooves whose interior-facing edges are rounded. These components may be located inside an incubator suitable to maintain desired environmental conditions and cleanliness.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/686,211, filed Aug. 25, 2017, and claims the benefit of U.S.Provisional Patent Application No. 62/380,414, filed Aug. 27, 2016, thedisclosures of which are both incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

Embodiments of the invention pertain to bioreactors.

BACKGROUND OF THE INVENTION

Bioreactors are used to expand a population of cells, such as stem cellsor other anchorage dependent cells. However, improvements are stilldesirable, such as in regard to ease of use, and automation, andreproducibility of procedures. It is also desirable to culture so as toproduce large numbers of cells, such as billions of cells if possible.

SUMMARY OF THE INVENTION

According to one embodiment of the invention, a bioreactor systemincludes a reservoir container for holding a liquid medium and amanifold assembly having an upper manifold and a lower manifold thatextends into the reservoir container. The bioreactor system furtherincludes a holder that is contained within the manifold assembly andholds a plurality of fiber assemblies that are suitable for cells togrow on, and a circulation system for causing the liquid medium to flowthrough the lower manifold, fiber assemblies and upper manifold. Theliquid medium flows through a sequence that comprises (a) one of thefiber assemblies that comprises a plurality of solid fibers that arejoined to other solid fibers at crossing points with other solid fibers,followed by (b) an open region in which the liquid medium flowsgenerally perpendicular to the one of the fiber assemblies through spacenot occupied by any solid object. This sequence repeats itself aplurality of times along a direction of flow of the liquid medium. Thefiber assemblies are held in the holder such that the fiber assembliesdo not touch any neighboring one of the fiber assemblies under staticconditions and do not touch any neighboring one of the fiber assembliesunder conditions of flow of the liquid medium during cell culture.

According to another embodiment of the invention, a bioreactor systemincludes a reservoir container for holding a liquid medium and amanifold assembly comprising an upper manifold and a lower manifold thatextends into the reservoir container. The bioreactor system furtherincludes a holder contained within the manifold assembly and holding aplurality of fiber assemblies that are suitable for cells to grow on,and a circulation system for causing the liquid medium to flow throughthe lower manifold, the fiber assemblies and the upper manifold. Aninternal cross-sectional dimension of the lower manifold below theholder, an internal cross-sectional dimension of the upper manifoldabove the holder, and an internal cross-sectional dimension of theholder are all substantially equal to each other. A side-to-sidedimension of the fiber assembly is larger than a corresponding insidedimension of the holder, and the side-to-side dimension of the fiberassembly is smaller than a corresponding inside dimension of themanifold assembly in which the holder resides.

According to another embodiment of the invention, a method of culturingcells includes a first step of providing a bioreactor, the bioreactorincluding a reservoir container for holding a liquid medium, a manifoldassembly having an upper manifold and a lower manifold that has a lowerend extending into the reservoir container, a holder contained withinthe manifold assembly and holding a plurality of fiber assemblies thatare suitable for cells to grow on, and a circulation system for causingthe liquid medium to flow through the lower manifold, the fiberassemblies and the upper manifold. The liquid medium flows through asequence that comprises (a) one of said fiber assemblies that comprisesa plurality of solid fibers that are joined to other solid fibers atcrossing points with other solid fibers, followed by (b) an open regionin which the liquid medium flows generally perpendicular to the one ofthe fiber assemblies through space not occupied by any solid object. Thesequence repeats itself a plurality of times along a direction of flowof the liquid medium. The fiber assemblies are held in the holder suchthat the fiber assemblies do not touch any neighboring one of the fiberassemblies under static conditions and do not touch any neighboring oneof the fiber assemblies under conditions of flow of the liquid mediumduring cell culture. An internal cross-sectional dimension of the lowermanifold below the holder, an internal cross-sectional dimension of theupper manifold above the holder, and an internal cross-sectionaldimension of the holder are all substantially equal to each other. Aside-to-side dimension of the fiber assembly is larger than acorresponding inside dimension of the holder, and the side-to-sidedimension of the fiber assembly is smaller than a corresponding insidedimension of the manifold assembly in which said holder resides. Themethod includes the further steps of seeding cells onto the fiberassemblies, operating the circulation system under conditionsappropriate for the cells to multiply into a plurality of culturedcells, and harvesting the cultured cells from the bioreactor.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described but are in no waylimited by the following illustrations.

FIG. 1A shows an overall layout of some of the major components of abioreactor of an embodiment of the invention.

FIG. 1B shows the components of FIG. 1A in cross-section.

FIG. 2A shows an assembly, in cross-section, comprising the lowermanifold and the upper manifold.

FIG. 2B shows the same assembly as FIG. 2A, with the upper manifoldhighlighted.

FIG. 2C shows the same assembly as FIG. 2A, with the lower manifoldhighlighted.

FIG. 3A is a three-dimensional view showing a screen holder, withoutscreens.

FIG. 3B is a three-dimensional view showing the screen holder of FIG.3A, exploded.

FIG. 3C is a three-dimensional view showing the screen holder withscreens in it. For clarity of illustration, one part of the screenholder is colored differently from the other part.

FIG. 3D is a three-dimensional view showing the screen holder of FIG. 3Cwith one part of the screen holder missing in order to better show thescreens.

FIG. 3E is a three-dimensional view showing the screen holder of FIG. 3Dwith some of the screens missing.

FIG. 4A is a view of the closure piece of the screen holder, showingrounded entrances of slots in the screen holder.

FIG. 4B is another view of the closure piece of the screen holder, froma different perspective.

FIG. 4C is a view of a local region of the closure piece.

FIG. 5A shows a screen made according to an embodiment of the invention.

FIG. 5B shows a closer view of a similar screen.

FIG. 5C illustrates spacing parameters of the screens such as those inFIGS. 5A and 5B.

FIG. 5D illustrates more specifically the placement of fibers within ascreen, and their staggering.

FIG. 6A illustrates a possible leakage flowpath.

FIG. 6B illustrates places where liquid medium is present during typicaloperation of the system.

FIG. 7A illustrates the three-sided piece that is part of the screenholder, so as to illustrate positioning slots.

FIG. 7B is a cutaway view illustrating the three-sided piece of FIG. 7A,together with a pusher accessory.

FIG. 7C illustrates the pusher accessory.

FIG. 7D illustrates a recess and boss for engagement of the screenholder with the upper manifold.

FIG. 8 illustrates details of seals.

FIG. 9 illustrates placement of two bioreactors of embodiments of theinvention within an incubator.

FIG. 10A illustrates flowpaths of various gases and liquids includinginto and out of the incubator and into and out of a reservoir containerand manifold assembly.

FIG. 10B illustrates a showerhead connected to the reservoir container.

FIG. 11 shows an overall layout of a bioreactor of another embodiment ofthe invention.

FIG. 12 shows an overall fluid flow path within the bioreactor.

FIG. 13 shows a reservoir base including several recesses.

FIG. 14 shows the riser conduit and nearby components.

FIG. 15 shows an exploded view of the rotor chamber and rotor.

FIGS. 16A-16C show various views of the rotor.

FIG. 17 shows details pertaining to the levels of surfaces of liquids inthe bioreactor.

FIG. 18 shows a filler structure inside the riser conduit.

FIG. 19A shows results of Computational Fluid Dynamics modeling of theoverall system at a first flowrate of liquid.

FIG. 19B shows results of Computational Fluid Dynamics modeling of theoverall system at a second flowrate of liquid.

FIG. 19C shows results of Computational Fluid Dynamics modeling of theoverall system at a third flowrate of liquid.

FIG. 20 shows results of Computational Fluid Dynamics modeling nearindividual screens. 10.

DETAILED DESCRIPTION OF THE INVENTION

One phenomenon that is important in cell culture is the limitingdistance for diffusion of nutrients and waste products during growth andmaintenance of cells and tissue. Tissue in living organisms organizesitself so that cells are never separated by more than a certain distancefrom a blood vessel or transport path. The maximum distance for cells tobe located away from a transport vessel such as a blood vessel is aboutseveral hundred microns. In the design of bioreactors, an importantconsideration is to provide a geometry in which cells are neverseparated from liquid medium by a distance greater than several hundredmicrons.

Another phenomenon that is relevant to isolated cells surrounded byliquid is motion of the cells through the liquid. The physicalproperties (density, viscosity) of a liquid culture medium typicallysimilar to the physical properties of water. Cells that are loose in aliquid culture medium are slightly denser than the density of the liquidculture medium that surrounds them, which causes the cells to fall orsink downward under the influence of gravity. If a cell is considered tobe a sphere moving in a viscous liquid, the situation is described byStokes' Law. In this situation, the settling velocity, V, is given byV=(ρ_(cell)−ρ_(fluid))*g*D ²/(18*μ)where D is the diameter of the sphere, (ρ_(cell)−ρ_(fluid)) is thedifference in density between the sphere and the fluid, g is theacceleration due to gravity, and p is the viscosity of the fluid. Thisequation is valid for conditions of laminar flow.

Another consideration in cell culture is to provide a gaseous atmospherein the incubator. which results dissolved gas in the cell culturemedium, containing sufficient oxygen and also containing carbon dioxidein a desired concentration of about 20% and 5% respectively, and havinga desired relative humidity such as about 95%. Yet another considerationis to provide a desired temperature such as approximately normal humanbody temperature, such as 37.0 C.

Another consideration in cell culture is the shear stress due to liquidthat may be flowing past the cells that are attached to or in theprocess of attaching to a substrate or cell culture scaffold. It isdesirable that such shear stress not exceed a certain value, so as notto dislodge the cells from the substrate or scaffold. It is believed tobe especially important that the shear stress be small during the earlystages of cell seeding in a bioreactor, when cells are still in theprocess of forming an attachment to the substrate or scaffold. Formesenchymal stem cells, it is believed to be desirable to keep the localshear stress under 0.1 Pa.

Herein, a first embodiment of the invention is described in which thecells are statically seeded onto the scaffolds that are in the form ofscreens, and the screens are then mounted inside a bioreactor, whereliquid medium perfuses through the screens generally perpendicular tothe screens.

Referring now to FIGS. 1A and 1B, there is illustrated a bioreactor 10of an embodiment of the invention. The bioreactor 10 may comprise areservoir container 60 suitable to hold a desired quantity of a liquidmedium. The bioreactor 10 may also comprise a reservoir cover 62corresponding to reservoir container 60. The reservoir cover 62 mayattach to the reservoir container 60 by a snap or other suitablefastener or attachment. The reservoir cover 62 may have an opening 64therethrough suitable to receive a flow structure extending through theopening 64 and suitable to extend below a surface of liquid medium inthe reservoir container 60. The flow structure may comprise, asillustrated, various pieces that fit together and may receive or enclosea screen holder 200. The screen holder 200 in turn may hold screens 300upon which cells reside and grow. The array of screens 300 can bereferred to as a culture structure.

Upper Manifold and Lower Manifold

Referring more specifically to FIGS. 2A, 2B and 2C, the flow structuremay comprise a manifold assembly that comprises lower manifold 82 and anupper manifold 86. Lower manifold 82 and an upper manifold 86 may fittogether with each other, and in combination may surround and enclosescreen holder 200. Lower manifold 82 may extend downward into reservoircontainer 60 sufficiently so that the bottom end of lower manifold 82may extend below a desired level of liquid medium that is stored in thereservoir container 60.

The interior of lower manifold 82 may be generally close-fitting withrespect to the exterior of screen holder 200. Lower manifold 82 may havea ledge 84. Screen holder 200 may rest upon ledge 84, but screen holdermay also be able to move slightly upward in the vertical direction.

Upper manifold 86 may be suitable to fit together with lower manifold82. Upper manifold 86 may define an internal passageway therethroughthat is in fluid communication with the interior of lower manifold 82and with the interior of screen holder 200 if screen holder 200 ispresent inside lower manifold 82. Upper manifold 86 may have an internalthat is of similar shape to the interior of lower manifold 82. Theinterior of upper manifold 86 and the interior of screen holder 200 maybe of similar or identical shape and dimensions so that changes ofcross-sectional flow area and changes of flow direction are small ornon-existent as flow goes through lower manifold 82 and then throughscreen holder 200 and then through upper manifold 86.

Upper manifold 86 may have, above the location where it interfaces withlower manifold 82, an upper end that is weir or overflow wall 112. Weir112 may be a generally horizontal edge defining a perimeter of thepassageway through upper manifold 86. When liquid is present and flowingin the system with an air space in the upper portion of upper manifold86, weir 112 may define a liquid level of liquid inside upper manifold86 and related components. External of weir 112 may be a moat 116 mayhave a bottom that is at a lower elevation than the top of weir 112.Moat 116 may be suitable to receive and contain liquid that overflowsweir 112. Moat 116 may further have a sump 118, which may be a localizeddepression that is smaller than moat 116 and is at a lower elevationthan other parts of moat 116. There may further be provided, in fluidcommunication with sump 118, an exit passageway by which liquid mediummay exit to other parts of a fluid circuit.

Upper manifold 86 may engage with lower manifold 82 in a slideablemanner that is guided by a feature of upper manifold 86 being parallelto, of similar shape, and of similar dimensions but allowing enoughlooseness for motion, with respect to a corresponding feature of lowermanifold 82. As illustrated, the feature of upper manifold 86 is locatedoutwardly with respect to the feature of lower manifold 82. Uppermanifold 86 may have a contact with lower manifold 82 that limits themotion of upper manifold 86 toward lower manifold 82, forming a stopconstraint. Alternatively, it is possible that motion of upper manifold86 and lower manifold 82 toward each other could be stopped when anappropriate surface (which may be flat) of upper manifold 86 makescontact with an upper surface of screen holder 200, and correspondinglya lower surface of screen holder 200 makes contact with an appropriatesurface (which may be flat) of lower manifold 82.

There may also be provided a top cover 130 that covers the top of uppermanifold 86. Top cover 130 may be at least approximately a flat plate.If desired, top cover 130 may have stiffening ribs. Top cover 130 may bepermanently joined to upper manifold 86 and, there may also be provideda seal between top cover 130 and upper manifold 86. It wouldalternatively be possible if desired to provide some form of fastenerconnecting top cover 130 and upper manifold 86.

As illustrated, opening 64 through reservoir cover 62 may have a raisededge or lip 68 surrounding it, and lower manifold 82 may have a flange83 surrounding lower manifold 82 on its exterior. Flange 83 may restupon raised edge or lip 68 to define a spatial relationship betweenlower manifold 82 and reservoir cover 62. Raised edge or lip 68 mayprovide that if any condensation of liquid water occurs on reservoircover 62, and if such condensate is not sterile, such condensate can beprevented from dripping into reservoir container 60. Such condensate maybe confined to the top (external) surface of reservoir cover 62. Variousdesigns of such lip are possible.

Seals and Clamps

Lower manifold 82 may have a lower manifold seal groove 92 that issuitable to contain a compressible seal 94 for creating a seal betweenlower manifold 82 and upper manifold 86. This compressible seal 94 isillustrated as having a shape, in cross-section, that is rectangularwith deformable triangular fingers on one surface. Of course, othercross-sectional shapes are also possible. Preload between upper manifold86 and lower manifold 82 may be created by clamping devices, which maybe over-center type clamping devices. Two such clamping devices 97A, 97Bare illustrated. When the described stop situation is reached based onposition of upper manifold 86 and lower manifold 82 relative to eachother, compressible seal 94 may occupy a compressed configuration.

Upper manifold 86 may have an upper manifold seal groove 96 that issuitable for holding a compressible seal member 98 for creating a sealbetween upper manifold 86 and top cover 130. This compressible sealmember 98 is illustrated as being a typical O-ring. Preload between topcover 130 and upper manifold 86 may be created by snaps (notillustrated), clamps (not illustrated) or any other appropriate designfeatures. Another possibility is that top cover 130 can be permanentlyjoined to upper manifold 86 such as by adhesive.

Screen Holder

In an embodiment of the invention, the screens 300 may be held by ascreen holder 200. The screen holder 200 is illustrated in FIGS. 3A-3Eand FIGS. 4A-4C. Screen holder 200 may hold a desired number of screensin a desired spaced position and orientation. The screen holder 200illustrated in FIGS. 3A-3E and FIGS. 4A-4C holds 15 screens 300. It maybe desirable that the number of screens be not much larger thanapproximately 15 in order to avoid creating gradients of oxygenconcentration in the liquid medium during operation of the bioreactor.

In general, screen holder 200 may surround a hollow interior space andmay be any of various shapes such as round, rectangular, etc. Asillustrated, the screen holder 200 (on both its both interior and itsexterior) has the general shape of a rectangle with rounded corners. Thescreens 300 held by the illustrated screen holder 200 are rectangular.For sake of convenience in description, screen holder 200 is describedhere using directional designations such as front and back, sides, andhorizontal and vertical, which generally correspond to their orientationor position as illustrated in the assembled bioreactor 10. However, itmay be understood that these directional designations are somewhatarbitrary.

When screens 300 are in place in screen holder 200, there may be adesired amount of space in the vertical direction between a screen 300and its nearest neighbor screen. In the bioreactor 10 as illustrated,the flow of the liquid medium can be generally perpendicular to the flatsurface of screens 300, flowing through the open spaces in the screen300.

A cell culture region could contain, for example, approximately 15 suchscreens 300 spaced apart from each other by a sufficient distance sothat the screens do not touch each other and some sideways flow ofliquid medium is possible if necessary.

Screen holder 200 could be of a hollow simple shape some of which formsa complete perimeter, with the shape being a shape such as round orrectangular (possibly with rounded corners). In general, it is possiblethat screen holder 200 could be made as a single piece, such as by anadditive manufacturing process. However, perhaps more typically screenholder 200 could be made out of two pieces, such as two pieces of moldedplastic, that join together with each other. It is illustrated thatscreen holder 200 is made of two inter-engaging pieces. The screenholder 200 may comprise a three-sided piece 220 and a closure piece 240that is engageable with the three-sided piece 220. The three-sided piece220 may comprise, in sequence, a first side segment 222A, a rear segment222B and a second side segment 222C opposed to the first side segment222A. The closure piece 240 may comprise at least one segment, which maybe considered a front segment 242B. As illustrated, the closure piece240 may additionally comprise a first side segment 242A and a secondside segment 242C. Thus, in sequence, there is a first side segment242A, a front segment 242B and a second side segment 242C. The firstside segment 242A may be engageable with the first side segment 222A,and the second side segment 242C may be engageable with the second sidesegment 222C. However, it is also possible that the closure piece 240might comprise only a front segment 242B. As illustrated, front segment242B and rear segment 222B are substantially parallel to each other, andfirst side segment 222A and second side segment 222C are substantiallyparallel to each other and first side segment 242A and second sidesegment 242C are substantially parallel to each other. However, otherspatial relationships are also possible.

The engagement features may comprise deformable tabs 800, and furthermay comprise an opening 224A in first side segment 222A and a similaropening 224C in second side segment 222C, with the openings 224A and224C being appropriately designed to engage with tabs 800. The tabs 800may be elastically deformable between an engagement position asillustrated, and a release position in which the deformable tabs 800 arebent inward sufficiently to create disengagement. The tabs 800 maycomprise living hinges, and it is further possible that elasticityelsewhere in the closure piece 240 may also contribute to changes ofdimension or shape so as to permit or assist in achieving disengagement.

Screen holder 200 may have an upper edge and a lower edge that may beflat, and which may be parallel to each other.

Slots and Grooves in Screen Bolder, and Rounded Edges Near Slots andGrooves

The screen holder 200 may comprise various grooves and slots that definelocations of screens 300 and provide mechanical support for screens 300.As illustrated, screen holder 200 is able to hold 15 of the screens 300,but of course other numbers of screens 300 are also possible. Thespacing distance between screens 300 may be chosen from a combination ofconsiderations such as patterns of fluid flow between screens 300, anddesired overall packing density of cells in the cell culture region ofbioreactor 10.

If screen holder 200 were made as a single piece (similar to what isillustrated but with the three-sided piece 220 and the closure piece 240joined together), it would be possible to insert and remove screens 200into or from screen holder 200. This can be done from a sidewaysdirection.

In the screen holder 200 as described and illustrated, it is possible toinsert and remove screens 300 into or from screen holder 200 whenthree-sided piece 220 and closure piece 240 are already assembled toeach other, without disassembling those pieces from each other. This canbe done from a sideways direction. As illustrated, first side segment222A has grooves 170, and second side segment 222C has grooves 170. Rearsegment 222B has grooves 170. Front segment 242B has slots through whichscreens 300 may pass without disassembly of screen holder 200. Thegrooves 170 may be substantially coplanar with each other and may havesimilar or identical dimensions to each other in the vertical direction.Slots 180 may be parallel to or coplanar with at least some of grooves170. The vertical dimensions and elevations of slots 180 may beidentical to those of grooves 170, although it is sufficient if thevertical dimensions and elevations are simply generally similar to eachother. These various features may combine and interact so that whenscreen holder 200 is assembled, it is possible for screens 300 to beslid into place through slot 180 and to be supported by grooves 170, aswell as by slot 180. The desired spacing or separation between screens300 is maintained by the material of screen holder 200 near grooves 170,and by the material of screen holder 200 (ligaments) that exist betweenslots 180.

As illustrated, closure piece 240 has three sides, with two of the sides242A, 242C being short. One side wall of screen holder 200 comprisesboth first side segment 222A and first side segment 242A, and similarlythe other side wall comprises both second side segment 222C and secondside segment 242C. As illustrated, even tabs 800 contain grooves thatare continuous with grooves 170 in the corresponding adjacent sidesegments. However, it can be understood that various other designs arealso possible.

As illustrated, grooves 170 in first side segment 222A and first sidesegment 242A are substantially sharp-edged. The same is true for grooves170 in second side segment 222C and second side segment 242C. However,it can be understood that other geometries are also possible.

On a front portion of the screen holder 200, the separators or ligamentsof remaining material may be such that there are rounded edges facegenerally exteriorly of the screen holder 200. This can be helpful inguiding screens 300 into slots 180 during initial insertion of thescreens 300 into slots 180. The rounded edges may be hemicylindrical.Such rounded edges can also be helpful in guiding screens 300 into slots180 if it happens that any of the screens 300 are not perfectly planar,such as being slightly warped out-of-plane.

At a rear portion of the screen holder 200, separators betweenindividual screens 300 may be such that there are rounded edges facegenerally toward the interior of the screen holder 200. This can behelpful in guiding screens 300 into desired positions when screens 300are being inserted into screen holder 200, such as near the end of theinsertion process. The rounded edges may be hemicylindrical. The roundededges may be hemicylindrical. Such rounded edges can also be helpful inguiding screens 300 into grooves 170 if it happens that any of thescreens 300 are not perfectly planar, such as being slightly warpedout-of-plane.

The separators at the front portion of the screen holder 200 may defineslots through which the screens 300 may pass. The separators at the rearportion of the screen holder 200 may define grooves into which thescreens 300 may enter but through which it is impossible for screens 300to pass.

In the case of both the rounded edges of the slots 180 and the roundededges of grooves 170, a hemicylindrical curvature is only one of variouspossible curvatures. A fillet having some other desired radius is alsopossible. Such radius could be either less than or greater than theradius of the hemicylinder. Other shapes of curves are also possible.Variation of curvature along the length of the slot 180 or groove 170 isalso possible.

Design of Screens

In embodiments of the invention, and referring now to FIGS. 5A-SD, thebioreactor 10 may have, within the culture region, an array of screens300, which serve as tissue scaffolds for cells such asanchorage-dependent cells to grow upon. The individual screens 300themselves may comprise a number of layers of fibers 400, with thefibers 400 having orientations that are perpendicular to theorientations of fibers in an adjacent layer. In a particular embodimentof the invention, the number of layers of such fibers 400 in anindividual screen 300 may, for example, be four or five or six layers.

Typical dimensional parameters of such screens 300 could be a fiberdiameter of 150 microns and a fiber spacing (as defined in FIG. 5C) of200 microns, with that dimension referring to the distance from an edgeof one fiber to the nearest edge of the nearest fiber in the same plane.Fibers 400 in one layer may be staggered relative to fibers 400 that areparallel to them and are located in a different layer of the screen 300,or alternatively there does not have to be staggering. The fibers 400may be spaced apart from each other by appropriate distances such thatduring initial cell seeding, the cells deposit on fibers 400 and do nottouch cells on adjacent fibers 400. However, the spacing of the fibers400 may be such that after some number of cell multiplications, cellsgrowing on nearby fibers 400 may contact or grow against each other (asituation known as confluence).

More particularly, the fibers 400 may exist in two mutuallyperpendicular directions and may be staggered in both of those twomutually perpendicular directions. This is further illustrated in FIG.5D. Each screen may comprise a plurality of fibers forming a first layerhaving parallel fibers oriented in a first direction and may comprise asecond layer having a plurality of parallel fibers oriented in a seconddirection that is perpendicular to the first direction, the fibers inthe second layer being joined to the fibers in the first layer. At leastsome of the screens comprise a plurality of fibers, and within anindividual one of the screens, the fibers are arranged in sequence in atleast first, second, third and fourth layers, the fibers within each ofthe layers being generally parallel to each other, wherein fibers in thefirst layer are generally parallel to fibers in the third layer andfibers in the second layer are generally parallel to fibers in thefourth layer, and wherein when viewed perpendicular to a flat surface ofthe screen, fibers of the third layer are located non-aligned withfibers of the first layer and fibers of the fourth layer are locatednon-aligned with fibers of the second layer. More specifically, withrespect to this viewing direction, the fibers may be located midwaybetween the parallel fibers that they are not aligned with. Suchconfiguration can help to prevent cells from falling through the screen,especially during initial seeding, while still providing space forliquid medium to occupy and flow through, and providing space for cellsto grow into as the cells multiply.

A screen 300 may have a particular number of layers, such as fourlayers, with media available at both surfaces of the four-layerconstruct, for contacting cells, and media also available in openingsthat exist between layers. It is believed that all of the features ofthis situation more closely resemble what occurs in natural tissues,which can be termed a three-dimensional environment. It is believed thatthe three-dimensional situation of embodiments of the invention is moreconducive to cell multiplication and expansion than is a two-dimensionalenvironment. However, it is not wished to be limited to thisexplanation.

The screens 300 may be formed by a programmed deposition of heatedfilaments of polymer, similar to what is described in commonly-ownedU.S. Pat. No. 8,463,418. The polymer may be a suitable biocompatiblepolymer such as polystyrene. The screen 300 as described is non-woven.Alternatively, screen 300 may be woven if desired. The overall shape ofthe screen 300 may be flat and rectangular.

In some prior art cell culturing techniques such as Petri dishes, alayer of cells grows on a flat surface and experiences an environmentthat is essentially two-dimensional. Even if there is adequate supply ofnutrients and removal of waste products in this situation, such atwo-dimensional environment is by inherently different from theenvironment in which cells naturally grow, which is a three-dimensionalenvironment.

In embodiments of the invention, cells seed by attaching onto individualfibers of the screen. The fiber-to-fiber dimension of the screen may belarge enough that when isolated cells initially attach to the fibers, atleast some of the cells generally do not touch other cells. After cellshave multiplied, and perhaps created several layers of cells whereinitially only one layer of cells was attached to a fiber, it ispossible that the outermost cells may still be independent of the nextfiber or it is possible that some of the new cells may touch other cellsthat are attached to other fibers, i.e., some bridging of fibers mayoccur (referred to as confluence). In embodiments of the invention, ascreen may have a particular number of layers of fibers, such as fourlayers. with media available at both surfaces of the four-layerconstruct, for contacting cells. At any rate, it is believed that all ofthe features of this situation more closely resemble what occurs innatural tissues, which can be termed a three-dimensional environment. Itis believed that the three-dimensional situation of embodiments of theinvention is more conducive to cell multiplication and expansion than isa two-dimensional environment. However, it is not wished to be limitedto this explanation.

A cell culture region could contain, for example, approximately 10 to 15such scaffolds spaced apart from each other by a sufficient distance sothat the screens do not touch each other and liquid can flow between thescaffolds.

In terms of biological parameters, a screen used with an embodiment ofthe invention may have an area (length dimension*width dimension) ofabout 7000 mm 2, and the portion of that screen exposed to perfusion(excluding the edges that are in grooves or slots) may have an area ofabout 6300 mm 2. This screen may be formed of four layers of fibers,with the layers alternating in direction as described elsewhere herein.On such a screen there may be deposited an initial seeding of about800,000 cells, so that if 12 screens are used, the population of seededcells is 9.6 million cells. At the end of culturing, that cellpopulation has expanded to about 250 million cells. Flow of liquid mediaspeed based on the empty space in the screen, for dynamic culturing, canbe approximately 2 cm/min. It is believed that a flow velocity of 1.6mm/sec, based on the empty space for passage of liquid through thescreen, will produce a maximum shear stress, at the edge of a fiber inthe screen, of 0.1 Pa, and it is believed to be desirable that formesenchymal stem cells the shear stress should stay below this value.

Dimensional Considerations Affecting Flow

In embodiments of the invention, the general direction of flow of theliquid medium may be perpendicular to and through the screens 300, whichas illustrated is vertically upward.

In embodiments of the invention, cells seed by attaching onto individualfibers 400 of the screen 300. The fiber-to-fiber dimension of the screen300 may be large enough that when isolated cells initially attach to thefibers 400, they generally do not touch other cells. After cells havemultiplied, and perhaps created several layers of cells attached to afiber 400 where initially only one layer of cells was attached to afiber 400, it is possible that the outermost cells may still be out ofcontact with cells that are attached to the nearest-neighbor fiber 400,or it is possible that some of the new cells may touch other cells thatare attached to other fibers 400, i.e., some bridging between fibers(confluence) may occur. Even if confluence does not occur, afteradditional cells have grown, the open space for passage of liquid mediumthrough the screen is smaller than it was at the start of culturing.

In embodiments of the invention, the screens 300 may fit within grooves170 or slots 180 of screen holder 200 with a slight degree of looseness.This is so that screens 300 can be easily inserted into or removed fromthe grooves 170 and slots 180. For example, a total height of a screen300 may be 600 microns, while the vertical dimension of a groove 170 orslot 180 may be 1 millimeter. This leaves a gap, in the verticaldirection, between the screen 300 and the groove 170 or slot 180. In thehorizontal direction, in a side-to-side direction, the dimensions of thescreen 300 may be slightly smaller than the horizontal dimensionsdefined by the base of a groove 170 on one side of the screen holder 200to the base of the groove 170 on the other side of screen holder 200. Inthe horizontal direction, in the front-back direction, the location ofthe screen 300 may be determined by two limiting structures betweenwhich the screen 300 may fit with a slight degree of looseness. One ofthose structures may be the interior wall of the lower manifold 82. Theother structure may be the inward-facing extreme of boss 216. In thefront-back direction, the screen may be slightly smaller, at least atthe location of the boss 216, than the distance between the boss 216 andthe opposed inward-facing surface of lower manifold 82. Alternatively,it is also possible that some features of screen holder 200 could beinvolved in determining the position of screen 300.

The following forces can act on a horizontally-oriented screen duringflow through the screen: the weight of an individual screen whensubmerged in the liquid medium; and the drag force due to flow of theliquid medium (which can be approximated as the pressure differenceacross the screen holder, divided by the number of screens). This forcebalance will determine whether the screens sit on the lower edge of thegroove, or are pushed upward and pinned against the upper edge of thegroove, or float somewhat unstably in between those other two positions.At least one of these quantities, i.e., the pressure drop, can varyduring the course of culturing, as the screens become more crowded withcells.

At the same time, it is desired that most of the flow of liquid mediumgo through the openings in the screen 300.

In the described stack of screens 300 and within the described screenholder 200, there are various possible local flowpaths of the liquidmedium. In much of the screen 300, there is open space between fibers400 through which the liquid medium can flow. Prior to the seeding ofcells onto the screen 300, the dimensions of the minimum flow area for aparticular opening between fibers 400 can be approximated as theinter-fiber spacing distance by the inter-fiber spacing distance. Also,prior to the seeding of cells, when the screen is mounted into thescreen holder 200, there is a possible flow path at the edges of thescreen 300. Such flowpath is illustrated in FIG. 6A. Flow in suchflowpath may be influenced by local dimensions such as the inter-fiberspacing distance, and the gap in a horizontal direction between thescreen 300 and the screen holder 200, and the gap in a verticaldirection between the screen 300 and the screen holder 200. However, forsimplicity of discussion, it can be assumed that the area for leakageflow, for one space between fibers 400, is the inter-fiber spacingdistance by the inter-fiber spacing distance.

It may be of interest to compare the desired flowrate to the leakageflowrate, in the form of a ratio of those two quantities. If the screenexternal dimensions are assumed for simplicity to be square with a sidelength of L, the number of cells along the perimeter would be 4*L/Δ,where Δ is the inter-fiber spacing distance. The total flow area of allsuch leakage paths then would be (4*L/Δ)*Δ², or 4*L*Δ. In the samesituation, for desired flow, the total flow area through the screen isL²*Δ². The ratio of desired flow area to leakage flow area isL²*Δ²/(4*L*Δ), or L/(4*Δ), or 0.25*(L/Δ). For the desirable goal ofhaving most of the flow go through the openings in the screens 300rather than through leakage paths, this incentivizes having a screen 300having many cells within the length of a side of the assumed squareshape. For example, the system may be designed so that a leakage pathcomprising a gap between an individual one of said screens and saidscreen holder, has a cross-sectional flow area that is less than 10%, orless than 2%, of a total flow area through all passageways through saidindividual one of said screens.

A further level of detail is to assume that the open spaces in the mainpart of the screen 300 become smaller as time goes on and the cellpopulation increases. It can be assumed that they come to a value thatmay be called Δ_(crowded). It is then necessary to make an assumptionabout the open spaces at the edge of the screen 300. It may be assumedthat the open spaces at the edge do not change their dimension, andtheir dimension remains at what may be called Δ_(edge). Then, the aboveratio of flow areas becomes L²*Δ_(crowded) ²/(4*L*Δ_(edge)), or0.25*L*Δ_(crowded) ²/Δ_(edge). This is of course subject to assumptionsespecially concerning the open dimension at the edge, i.e., Δ_(edge),but in general as Δ_(crowded) becomes smaller, the ratio of flow areafor desired perfusion flow, divided by flow area for leakage flow canbecome smaller than it was for the empty screen 300.

Another consideration affecting the design of a bioreactor may be thestiffness of the screens 300 against bending. When screens 300 are inthe bioreactor and are supported by screen holder 200, they aresubjected to gravity (either with or without buoyancy effects dependingon whether liquid medium is or is not present), and they also aresubjected to flow forces if the liquid medium is moving. Any such forcescould deform the screen 300 out-of-plane. It is desirable that thescreens 300 not deform so much that they touch each other or come loosefrom screen support 200. Stiffness of a screen 300 can be characterizedby its deformation in bending in a simple bending geometry in which twoopposed edges are supported as simple supports and the other two edgesare unsupported. For the described screens, which had a dimension alongthe direction of bending of about 100 mm, and a transverse dimension ofabout 60 mm, a weight of 14.5 grams applied at the center of the spancaused a deflection of 3.1 mm. The screen may be designed to be at leastas stiff in bending as the just-described bending stiffness. When thescreen is supported at four edges rather than the just-described twoedges, it will deflect less than in the just-described measurement.

Start-Up Procedure

In an embodiment of the invention, a start-up procedure can be asdescribed here. At the start of use of the system, the reservoircontainer 60 can contain liquid medium up to a desired liquid level. Theliquid level may be above the elevation of the bottom of lower manifold82, and the amount of liquid contained may be sufficient for desiredoperations. Pump 140 may then be operated so as to withdraw fluid frommoat 116. Pump 140 may be a positive displacement pump such as aperistaltic pump, capable of moving either liquid or gas or acombination thereof. Initially, pump will remove air from the regionincluding upper manifold 86. This removal of air will cause the level ofliquid medium to rise in lower manifold 82, into screen holder 300, andthen upper manifold 86. Eventually the liquid level will reach the levelof weir 112 and then begin to overflow weir 112 into moat 116. Liquidwill then flow into sump 118. Pump 140 withdraws fluid from sump 118. Itis possible that at some point during start-up, pump 140 will withdraw amixture of liquid and gas from sump 118, but that is all right.Eventually there will be reached a steady state in which a substantiallyconstant volume of gas remains in the region over upper manifold 86 andmoat 116. As long as pump 140 continues to run, there will be a flowrateof liquid over weir (overflow wall) 112 into moat 116 and sump 118 andtowards pump 140. It is believed that the level of liquid in moat 116will be fairly close to the bottom of moat 116. This is illustrated inFIG. 6B. The liquid is indicated in FIG. 6B by the dotted-hatchedpattern.

Pusher Accessory and Slots for Positioning of Screens

Referring now to FIG. 7 , in embodiments of the invention, as describedherein, the screen holder 200 may comprise positioning slots 390 thatcan be used for pushing screens 300 into a desired position or fordefining positions of screens 300. Such positioning slots 390 may begenerally vertical with respect to overall directions of the screenholder 200 and bioreactor 10. Positioning slots 390 may intersect otherslots and grooves that are provided in screen holder 200.

A pusher 400 may also be provided as an accessory (for use before orafter actual culturing). It is possible that the pusher 400 can be usedon one side of the screen holder 200 for the purpose of pushing screens300 into screen holder 200 until they contact a stop such as the base ofgroove 170. It is also possible that pusher 400 may be used on anopposite side of screen holder 200 for the purpose of pushing screens300 out of screen holder 200, such as when culturing is completed or itis desired to remove screens 300. Positioning slots 390 on one side ofscreen holder 200 may have identical or similar dimensions and spacingas positioning slots 390 on another side of screen holder 200, whichwould enable a single pusher 400 to be used for pushing in bothdirections.

In order to facilitate the described pushing, pusher 400 may havecertain dimensional relationships with appropriate features of screenholder 200. Bosses 420 on pusher 400 may be dimensioned, and may bespaced appropriately with respect to each other, so that they can fitinto positioning slots 390 of screen holder 200. The height of thebosses 420 on pusher 400 may be sufficiently large so that the screens300 may be pushed to a desired extent.

Features that Affect Engagement Between Screen Holder and Lower Manifold

It is further possible that there may be provided inter-engagingfeatures that constrain or guide the inter-engagement between screenholder 200 and lower manifold 82. Such features may be referred to as akey and a keyway. As illustrated, screen holder 200 has a recess 210,and lower manifold 82 has a boss 216. Of course, the opposite is alsopossible, i.e., a boss on screen holder 200 and a recess on lowermanifold 82. If screen holder 200 has some degree of symmetry, such asbeing of a rectangular cross-sectional shape, the key and keyway maylimit the number of ways in which screen holder 200 and lower manifold82 can be assembled together. This arrangement may serve to control thepositioning of screens 300 when screen holder 200 is assembled with therest of bioreactor 10.

There are some dimensional considerations in regard to insertion ofscreen holder 200 into lower manifold 82. For example, the width (in ahorizontal direction) of boss 216 may be less than the width of recess210.

If desired, the portions of the boss 216 or the recess 210, or both,that engage each other initially upon insertion of the screen holder 200into lower manifold 82, may be provided with rounding features at one oftheir ends (at the top of the feature on the lower manifold 82, or atthe bottom of the feature on the screen holder 200, or both), so as toguide the initial engagement between screen holder 200 and lowermanifold 82. It is also possible that a rounded feature may be providedat other places at the bottom of screen holder 200, or at the top oflower manifold 82, or both, so as to guide the initial engagementbetween screen holder 200 and lower manifold 82.

Incubator

Referring now to FIG. 9 , the bioreactor 10 may comprise an incubator950. The incubator 950 may maintain within itself conditions that areconducive to cell growth. The incubator 950 may maintain a temperaturethat is a desired value that is close to physiological temperature. Theincubator 950 may also maintain an atmosphere having a desiredcomposition. The composition may have an oxygen concentration ofapproximately 20% and a carbon dioxide concentration of approximately5%. The incubator 950 may also maintain a desired relative humidity ofthe atmosphere inside the incubator 950. The incubator 950 may have afront door providing access to the interior of the incubator 950 forinstallation or removal of major components. The incubator 950 also mayhave penetrations or pass-through through various of its walls. Suchpass-through and penetrations are described here for a particulardesign, although it can be understood that other such arrangements arealso possible.

At a location outside the incubator there may be a pump 140 forcirculating or controlling the flow of liquid media. Pump 140 may be apositive displacement pump such as a peristaltic pump. There may beappropriate pass-throughs or penetrations for passage of such liquidsinto and out of the incubator 950 to and from the pump 140. Locating thepump 140 outside of the incubator may serve some specific purposes. Onesuch purpose is that the electronics in the pump 140 may have a limit onthe humidity to which they can be exposed, which may be lower than therelatively humid conditions that are usually maintained inside theincubator 950. Locating the pump 140 outside the incubator 950eliminates this concern. Additionally, if the pump 140 was locatedinside the incubator 950, it could generate heat when it was running,and that heat could affect the control of temperature inside theincubator 950. Appropriate controls and, if desired, automation, may beprovided for the pump and related systems.

The just-described parts may be suitable to be placed inside acontrolled-environment chamber that may be referred to as an incubator950. Incubator 950 may have controls to maintain within its interior adesired temperature, a desired atmospheric composition, a desired oxygenconcentration, a desired carbon dioxide concentration, a desiredhumidity, desired values of any other environmental property, or anycombination of these. The incubator 950 may have a door to permit thepassage therethrough of these components, and the door may be closeableand sealable. The incubator 950 may further have passthroughs, throughthe incubator wall or boundary, which may be separate from the door.Such passthroughs may permit the passage of liquid into and out of theinterior of incubator 950. The incubator 950 may further haveconnections through which oxygen or carbon dioxide may be supplied tothe interior of incubator 950. Pump 140 for circulating liquid mediummay be located external to the incubator 950. Passthroughs may providefor the passage of liquid medium into and out of the incubator 950. Theincubator may be suitable to maintain sterility or at least cleanlinessat and near the described components such as the assembly of manifolds82, 86 and the reservoir container 60.

Connected to overflow moat 116 may be an exit fitting. The exit fittingmay exit the moat such that at a portion of the interior open space ofthe fitting is at or below the level of the base of the moat 116. Fromexit fitting, there may be tubing or similar fluid-carrying means goingto the intake of the pump 140. Tubing may be capable of conducting fluid(either liquid or gas) from the moat 116 to the pump. Pump 140 is shownas being located outside the incubator 1950. This pump location isoptional, although advantages of it are discussed elsewhere herein.

Downstream of the pump 140, flow may proceed back into the incubator 950and to a showerhead 186. The showerhead 186 may be located insideincubator 950 at the top of the reservoir container 60. The showerhead186 may distribute the liquid medium in the form of drops that fall backinto the reservoir container 60. The atmosphere inside the incubator 950may have a desired concentration oxygen, and a desired concentration ofcarbon dioxide, and may also be temperature controlled. During thepassage of drops of liquid medium from the showerhead 186 through theatmosphere inside the reservoir container 60, the liquid drops mayexchange gas with the atmosphere inside the reservoir container 60, suchas absorbing carbon dioxide from the atmosphere inside the reservoircontainer 60. The drops may also thermally equilibrate with theatmosphere inside the reservoir container 60 if needed. The drops maythen collect in liquid region of reservoir container 60.

Flow of fluid through the entire described flow path may then berepeated.

Reference is now made to FIG. 10 , which illustrates various connectionsthrough the boundary of the incubator 950 and the reservoir container60. The bioreactor 10 may comprise connections for pump 140 to withdrawliquid medium and to re-introduce liquid medium. This provides dynamiccirculation or perfusion of the liquid medium. If the pump 140 islocated outside the incubator 950, the liquid medium can exit from thebioreactor 10, pass through the boundary of the incubator 950, passthrough the pump 140, pass again through the boundary of the incubator950, and re-enter bioreactor 10. More specifically, the liquid mediumcan exit from the sump 118 that is connected to moat 116, and canre-enter the reservoir container 60 through showerhead 186. Theatmosphere inside the incubator 950 and the reservoir container 60 maybe managed in several ways. CO2 may be supplied from a source outsidethe incubator 950 and, as illustrated, passes through a passthrough thatis a permanent part of the incubator 950, shown as being on the rear ofincubator 950. Air can pass from outside the incubator 950 into theinterior of incubator 950 through a passthrough that is shown on theleft side of incubator 950. This entering air may pass through a filter960, which may be located outside the incubator 950. Thus, theatmosphere inside the incubator 950 may be a mixture of air and CO2. Thegas space of reservoir container 60 may have an air extractionconnection by which gas from the gas space of reservoir container 60 ispulled out of reservoir container 60, through a passthrough through theboundary of incubator 950, and out to an airflow pump 150. Airflow pump150 may be a peristaltic pump, which may be located outside incubator950. Gas from the atmosphere inside incubator 950 enters the reservoircontainer 60 through a filter (not illustrated) on port 940. Insidereservoir container 60, the showerhead 186 entrains some of that gas.The showerhead 186 causes aeration and exposes the liquid medium to CO2and to oxygen. Cells obtain both CO2 and oxygen from the liquid mediumfor their metabolism and for the ATP cycle for energy transfer. Theshowerhead 186 as illustrated has nonuniform hole size distribution inorder to form a spray with the desired characteristics and distribution.The liquid enters from above the showerhead 186 near the center of thearray of holes. Holes that are more outwardly located are larger indimension than holes near where the liquid enters.

As illustrated, two of the passthroughs through the left wall of theincubator 950 are for liquid medium to go into and out of the incubator.There is also a passthrough through the wall of the incubator by whichan airflow pump, which may be a peristaltic pump, pulls atmosphere outfrom the gas space of the reservoir container 60. There is also apassthrough through which air from outside the incubator 950 enters theincubator interior, passing through a filter 960.

Embodiment Comprising a Rotor and Two Different Orientations of Flow

In general, there are at least two possible geometries of planarscaffolds and flow in bioreactors. The previously described embodimentinvolved flow through a screen generally perpendicular to the plane ofthe screen. Another possible geometry involves flow of liquid in adirection that is generally parallel to a screen. Each orientation hasrespective advantages and disadvantages.

In this next embodiment of the invention, a bioreactor 1010 may haveoverall components and an arrangement as illustrated in FIG. 11 .

The described components may be attached to a base or frame that definestheir locations and allows the apparatus to be carried as a unit.External to the apparatus there may also be provided a source of carbondioxide, such as a pressurized tank. Similarly, a source of oxygen maybe provided if desired.

Flow Path

Referring now to FIG. 12 , for an embodiment of the invention, there isshown a fluid flow arrangement for the bioreactor 1010. As illustrated,the direction of liquid flow past the cell culture scaffold may be in agenerally upward direction with respect to gravity. The flow can flow ina recirculating flow path. In an embodiment of the invention the liquidmedium is able to flow in a recirculation path from a reservoir, upwardthrough the scaffold, through a pump, through a showerhead, and back tothe reservoir.

The pump 1140 may be capable of self-priming by pumping gas through thepump initially so as to pull liquid up from the lower reservoir into thecell culture region. Thereafter, the pump 1140 may be able to moveliquid when it is desired and at whatever rate it is desired tocirculate liquid through the culture region. The pump 1140 may be apositive displacement pump such as a peristaltic pump.

The apparatus may comprise a reservoir container 1060, which may belocated generally at an elevation that is lower than the elevation ofvarious other components of the apparatus. The bottom of the reservoircontainer 1060 may be formed, at least in part, by a reservoir base 1400that is generally flat and horizontal having a reservoir base uppersurface 1410. The reservoir 1060 may further be defined by reservoirsidewalls and a reservoir lid. Furthermore, reservoir base 1400 may havesome features that are recessed below reservoir base upper surface 1410.

The reservoir 1060 may have therewithin a stirrer such as a magneticstirrer bar (not illustrated) that can be caused to rotate by anexternally applied magnetic field that rotates. The stirrer bar may bemore dense than the density of the liquid so that the stirrer bar maysink due to gravity and rest upon a surface of the reservoir base, moreparticularly a bottom surface of stirrer recess 1420. Alternativestirrer arrangements are also possible, such as a rotating rod andpaddle.

The stirrer bar could be rotated at an appropriate rotational speed tomaintain cells in suspension in the liquid so that the liquid drawn intothe scaffold contains appropriate cells.

Reservoir Base

The reservoir 1060 may be defined in part by reservoir base 1400.Referring now to FIG. 13 . reservoir base 400 may be generally flat andhorizontal but may have a pattern of recesses recessed into it.

Corresponding to the stirrer bar, there may be a stirrer recess 1420,recessed into reservoir base 1400. Stirrer recess 1420 may be, in itshorizontal dimensions, larger than the path that is swept out by thestirrer bar. At least some of the stirrer recess 1420 may beapproximately cylindrical and may have a bottom that is flat andhorizontal. The depth of the stirrer recess 1420 can be less than thevertical dimension of the stirrer bar, if desired.

There may further be provided a canal recess 1430 that may be generallystraight in a lengthwise direction. There may further be provided areservoir sump recess 1440. The canal recess 1430 may intersect with thestirrer recess 1420, and may intersect with the reservoir sump recess1440.

The floor surfaces of all of these recesses (stirrer recess 1420, canalrecess 1430, reservoir sump recess 1440) may be planar and may becoplanar with each other and may be generally horizontal. Places wherethe various recesses (stirrer recess 1420, canal recess 1430, reservoirsump recess 1440) intersect each other may be provided with roundedcorners to improve the smoothness of fluid flow. At various placesassociated with the recesses, there may also be provided internalcorners that are rounded.

There may also be provided a drain recess 1450 that may be in fluidcommunication with the canal recess 1430 and may extend to a lowerelevation than does the canal recess 1430. Fluid can be removed from thereservoir 1060 by putting a tube into the drain recess 1450 from aboveand suctioning fluid out.

Referring now to FIGS. 14-16C, there may also be provided a riserconduit 500 that may define a flow path leaving the reservoir 1060 in anupward direction. The riser conduit 1500 is illustrated as being ofrectangular cross-section, but it can be appreciated that othercross-sectional shapes are also possible. The riser conduit 1500 mayhave a riser conduit lower edge 1510 that is flat and horizontal. Firstof all, the riser conduit 1500 may be positioned such that the riserconduit lower edge 1510 is at a lower elevation than the elevation ofthe liquid level in the reservoir 1060. More particularly, the riserconduit 1500 may be positioned so that the riser conduit lower edge 1510is located at an elevation that is higher than the floor of thereservoir sump recess 1440, but is lower than the upper surface 1410 ofreservoir base 1400. It may also be provided that reservoir sump recess1440 has a boundary that is larger than and outside the outer perimeterof riser conduit 1500. This combination of dimensional relationships mayprovide an inflow pattern, for flow from the reservoir 1060 into riserconduit, that is distributed generally around the entire perimeter ofriser conduit 1500. Such a flow pattern preserves access to a very largefraction of the liquid in the reservoir 1060, while not creating “pinchpoints” that have excessively high local fluid velocities or local shearrates in the fluid flow.

Continuing upward along the general direction of riser conduit 1500,there may be rotor chamber 1600 and rotor 1700 that is containedgenerally within rotor chamber 1600. More detail about rotor 1700 isprovided elsewhere herein.

Rotor chamber 1600 may have therethrough a vertical passageway definedby the lower pass-through opening 1610 and the upper pass-throughopening 1620. Lower pass-through opening 1610 and upper pass-throughopening 1620 may have substantially identical internal cross-sectionalarea and dimensions. The internal cross-sectional area and dimensions oflower pass-through opening 1610 and upper pass-through opening 1620 mayat least approximately match those of the interior of riser conduit 1500and those of the passageway (described elsewhere herein) through therotor 1700.

Rotor chamber 1600 may rest upon or may be mounted upon the cover plateof reservoir 1060.

Near the top of rotor chamber 1600 the rotor chamber wall that touchesthe liquid may end at a top edge 1630 that is flat and horizontal. Thetop edge 1630 may act as a weir for liquid to flow over it. Outside ofthe wall may be an overflow moat 1640. The design of the overflow moat1640 and nearby features may be such that the fluid in the rotor chamber1640 has to flow upward, then over the top edge 1630 of the rotorchamber wall, then downward into the moat 1640. The moat 1640 may extendaround the entire perimeter of the rotor chamber 1600. During operationof the system, a gas pocket may be present above the top edge 1630.

Rotor chamber 1600 may be topped off by rotor chamber lid 1660, whichmay be removable. There may be a gap between the top edge 1630 and therotor chamber lid 660. The gap may be suitable for flow to flow throughthe gap and thereupon the flow may enter and be collected by overflowmoat 1640. It is believed, although it is not wished to be limited tothis explanation, that the use of overflow moat 1640 may help to improvethe uniformity of flow through the cell culture region and tissuescaffold. There may be an overflow moat 1640, and a corresponding sump,and showerhead as in another embodiment herein.

Rotor and Cell Culture Scaffold

Within rotor chamber 1600 there may be provided rotor 1700. Rotor 1700may be able to rotate, with respect to rotor chamber 1600, around anaxis of rotation. The axis of rotation may be horizontal, and may begenerally perpendicular to the direction of the riser conduit 1500.Rotor 1700 may be close-fitting within rotor chamber 600, while stillbeing free to rotate.

One end of the rotor 1700 may have or may have attached thereto a shaftto direct the angular position of the rotor 1700. The shaft may in turnbe operated by a motor. The motor may be located inside the incubator1950, or may be located outside the incubator 1950 with a penetrationfor the shaft to pass through the wall of the incubator 1950. The motormay be controlled by a computer or automation system.

The rotor 1700 may be able to rotate through various rotation positions.One position of the rotor may correspond to a full-open flow path forflow through the rotor interior and the tissue scaffold. Anotherposition of the rotor 1700 may be such that the flow path is blocked bythe generally solid portion of the rotor 1700.

With continued reference to FIGS. 14-16C, there is shown the rotor 1700and some associated parts. The rotor 1700 may be generally cylindricalexcept for the absence of material as described herein. The rotor mayhave two opposed rotor openings 1710, 1712 defining a passageway throughthe rotor 1700, and the rotor may have two remaining opposed sides 1714,1716 that are mostly-solid. Proceeding around the perimeter of the rotor1700, there may be open space; a generally solid portion that may becylindrical on its exterior; open space; and another generally solidportion that may be cylindrical on its exterior. On the exterior, thegenerally cylindrical shape may correspond to the internal cylindricalspace within rotor chamber 1600. The dimensions of the mostly-solidsides may be such as to completely block the riser conduit 1500 (exceptfor holes 1790) when the rotor 1700 is at an appropriate angularposition. Rotor ends may have internally facing grooves 1720. Thegrooves 1720 in the rotor 1700 may be dimensioned appropriately toreceive and support the edges of scaffolds or screens.

One of the rotor openings 1710 may have a bridge 1740 that spans therotor opening. The other of the rotor openings 1712 may have a removableclip 1760 that spans the rotor opening. The bridge 1740 and the clip1760 may be dimensioned so that they occupy only a small portion of theoverall length of the rotor 1700 so as not to create a substantialdisturbance in the flow of liquid. The bridge 1740 and the clip 1760 mayhave internal grooves that correspond to the internal grooves 1720 atthe ends of the rotor 1700. In combination, the grooves at one end ofthe rotor 1700, the grooves at the other end of the rotor 1700, thegrooves in the bridge 1740 and the grooves in the clip 1760 may allalign with each other and may cooperate with each other to support thescreens.

Within the open space in rotor 1700 there may be provided cell growthscaffold such as screens 1300 as is described elsewhere herein, and flowmay flow past or through the cell growth scaffold such as screens 1300.

In the solid part of rotor 1700 that has the generally cylindricalexterior, there may be provided a hole 1790 or a plurality of holes 1790therethrough on each of two opposed sides. The holes 1790 may align witheach other. The holes 1790 may allow mass transfer between the liquid inthe scaffold region interior of the rotor 1700 and the liquid in thereservoir during the time when the scaffolds are in a horizontalorientation. Even though when the scaffolds (screens) are horizontalthere might be no bulk flow of liquid, holes 1790 would still allow somemass transfer even if it is only by diffusion or some form of naturalconvection.

The open space through the rotor 1700 may roughly correspond, indimensions and cross-sectional area, to the internal dimensions andcross-sectional area of the riser conduit 1500. For example, thedimension from the inside of one of the generally solid portions of therotor 1700 to the other of the generally solid portions of the rotor1700 may be the same or approximately the same as the inside dimension,in the same direction, of the riser conduit 1500. The end-to-enddimension of the empty space of the rotor may be the same orapproximately the same as the inside dimension in the same direction, ofthe riser conduit 1500.

The open space through the rotor 1700 may be the space intended to beoccupied by an array of cell culture scaffolds 1300. Ends of the rotor1700 that face internally to the open space may comprise grooves 1720 orsimilar interfaces to hold a plurality of cell culture scaffolds. Asillustrated, the ends of the rotor 1700 each have grooves 1720 for 10individual screens 1300. An alternative design could have grooves forsome other number of screens 1300 as might be desired. The grooves 1720can support or guide or position the ends of the screens 1300 at theedges of the screens 1300. The grooves 1720 may be dimensionedappropriately for the thickness of each scaffold or screen 1300, and forthe intended spacing between the scaffolds or screens 1300. At anadditional location such as midway between the two ends of the rotor1700, there may be provided small auxiliary supports that also containgrooves 1720 to support or guide or position the screens or scaffolds1300 at their edges. For example, there may be provided bridge 1740,which may be connected to the rotor 1700, which may contain grooves1720. There may be provided a removable clip 1760 that is attachable toand detachable from rotor 1700, and which also may contain grooves 1720.

Design of scaffolds and screens could be as discussed elsewhere hereinin connection with another embodiment.

As illustrated, the rotor 1700 contains grooves 1720 appropriate to holdthe scaffolds 1300 for flow in a direction that is parallel to thesurfaces of the scaffolds 1300. Alternatively, it would be possible toprovide a rotor 1700 that holds the scaffolds 1300 in a position forflow through the scaffolds (perpendicular to the face of the scaffolds).The apparatus could be designed to be able to accept various differentrotors 1700, with different rotors 1700 having different designs as faras placement or orientation of the scaffolds 1300. It is possible thatwith different interchangeable rotors, one rotor could provide flowgenerally parallel to the surface of the scaffolds while anotherdifferent rotor could provide flow through the scaffolds, i.e.,generally perpendicular to the scaffolds.

Considerations Related to Volume and Efficient Use of Cells and Media

Referring now to FIG. 17 , various considerations may be built into thedesign and operation of the apparatus to contribute to the efficient useof the medium, which can be an expensive material, and to the efficientuse of cells, which tend to sink under the influence of gravity. Thelower edge 1510 of riser conduit 1500 may extend submerged beneath thesurface of the liquid in the reservoir 1060, or specifically may extendsubmerged beneath the surface of liquid in reservoir sump recess 1440,both prior to operation and also at all times during operation. This maycontribute to the ability to suction fluid upward. In order to be ableto suction liquid upward into the riser conduit 1500, it is preferableto avoid having any air travel under the lower edge 1510 of the riserconduit 1500.

The available volume of the reservoir 1060 may be defined as the volumeof liquid present in the reservoir 1060, consistent with the requirementthat the liquid level is lower than the showerhead and lower than thestopper in the fill port, and with the liquid level in the riser conduitbeing the same as the liquid level in the reservoir 1060 outside theriser conduit 1500 when the apparatus is equilibrated and not operating.The available volume of the reservoir 1060 may be sufficient so thatwhen the reservoir 1060 contains an appropriate amount of liquid that isless than the available geometric volume of reservoir 1060, even whenthe rotor chamber 1600, the moat 1640, and various tubing are full ofliquid, the reservoir 1060 is still not empty or at least reservoir sumprecess 1440 is not empty. More specifically, at these conditions, theliquid level in the reservoir 1060 or reservoir sump recess 1440 isstill at a higher elevation than the lower edge 1510 of riser conduit1500.

The volume of the reservoir 1060 may be sufficient to fill all of thesejust-described spaces with liquid in the absence of screens beingpresent in the rotor 1700. Alternatively, the volumes of variouscomponents may be calculated, and may be corrected for the amount ofspace occupied by the screens of the cell culture scaffold, and thevolume of the reservoir may be sufficient to fill all of thesejust-described spaces when scaffolds are present. The volume of liquidthat is actually loaded into reservoir 1060 may be measured outsufficiently accurately so that when the empty spaces such as riserconduit 1500, rotor chamber 1600, moat 1640 and various tubing are fullof liquid, sufficient to reach the top of wall 1630 or at least to coverthe scaffolds with liquid, the liquid level in reservoir 1060 may be inthe reservoir sump recess 1440 but higher than the lower end of 1510 ofriser conduit 1500.

It has been illustrated that the upper surface 1410 of reservoir base1400 is flat and horizontal. Alternatively, it is possible that someslight slope or funnel shape could be provided to help drain liquid orto help liquid reach the reservoir sump recess 1440.

It can be noted that the pump 1140, such as a peristaltic pump, isdownstream of the cell culture chamber, i.e., rotor chamber 1600. Thismeans that during seeding of the scaffold, cells can reach the scaffolddirectly from reservoir 1060 without having to pass through the pump.Passage of cells through the pump could conceivably damage the cellsundesirably. However, it is anticipated that during seeding, most of thecells that occupy the region of the cell scaffold will depositthemselves on the scaffold. It is anticipated that only a small fractionof the cells will fail to seed and will exit the rotor chamber 1600 andpass through the pump 1140. If, upon passage through the pump 1140,there is damage to those cells that failed to attach to the scaffold,those cells should be few in number anyway, so possible damage to thoseunattached cells should not have much overall significance in terms ofthe efficiency of using cells.

It is possible that the direction of rotation of the stirrer bar can besuch that it urges flow of liquid that is in canal recess 1430 towardriser conduit 1500. As such, it would thereby provide some amount ofuseful pump-like action, especially when the liquid level in reservoir1060 is low, in addition to stirring action.

Filler Structure Inside Riser

Referring now to FIG. 18 , in an embodiment of the invention, there maybe provided a filler structure 1100 inside the riser conduit 1500.

The filler structure 1100 may usefully occupy some portion of the volumeinside riser conduit 1500 so that that portion of the volume does nothave to be occupied by media liquid. Because media liquid is expensive,and liquid media that is in the riser is does not contribute to cellseeding or culture, the filler structure 1100 may reduce such amount ofmedia that does not serve a useful purpose.

The filler structure 1100 may have a streamlined shape so as to receiveflow of media that enters at the bottom of the riser at the riser sump,and direct and distribute that flow upward toward the cell cultureregion. Accordingly, the upper end of the filler structure 1100 may bepointed, pointing vertically upward. The lower end of the fillerstructure 1100 may be such as to receive flow that has a horizontalcomponent of velocity coming from the reservoir sump recess 1440, andredirect that flow upwardly.

It is also possible to use a filler structure resembling fillerstructure 1100 in other embodiments of the invention. Even if this isnot done for the purpose of conserving the amount of liquid medium, itcould be beneficial for improving the flow patterns, such as improvingthe uniformity of velocity distribution, of flow entering the array ofscreens.

At a location outside the incubator 1950 may be a plurality of fluidcontainers. Such fluid containers may include a waste liquid bottle; aretrieval bottle; a media storage bottle; a PBS (phosphate bufferedsaline) bottle; and a tryp-LE E bottle. There may be appropriatepass-throughs or penetrations for passage of such liquids into and outof the incubator 950. Appropriate valves may also be provided forcontrolling the flow of such liquids. At this location, or generally atwhatever location is desired, there may also be provided a motor capableof rotating a cell culture component such as a rotor, as is describedelsewhere herein.

Impactor

In embodiments of the invention, there may also be provided an actuatoror impactor (not illustrated) that may be suitable to direct motion ator deliver an impact to the apparatus inside the incubator 1950, such asthe exterior of the rotor chamber. The vibration caused by the impactorstriking a component near the cell culture screens may dislodge cellsand may help in harvesting the cells after expansion. In the illustratedembodiment, the impactor could be located at the rear of the incubator1950. There may be an appropriate pass-through or penetration for theimpactor. This impacting may be done in conjunction with a chemicaltreatment such as exposing the cell population to trypsin.

The apparatus may also comprise automatic controls appropriate tooperate the described components.

Methods of Operation

In embodiments of the invention, seeding and growth of the cells ontothe scaffold may be performed by various protocols.

During seeding, the liquid in the reservoir 1060 could contain cells tobe seeded. During cell expansion, the liquid in the reservoir 1060 doesnot need to contain cells, but it could contain nutrients that areconducive to the growth and multiplication of cells.

It is contemplated that during the seeding stage, the cells/mediamixture will essentially not go through the pump 1140. At the start ofthe automatic seeding process, the cells and media are in the reservoir1060. The media volume at this time could be enough to fill the riserand the cell culture region, but not much more than that. When the pump1140 starts, this cells/media mixture will be suctioned up into the cellculture region. It is only necessary to suction up enough media to coverthe cell culture scaffold (screens) with liquid. It is unnecessary tofill the moat 1640 with liquid, and it is unnecessary to suction liquidfar enough for liquid to enter into the tubes that go from the moat 1640to the pump 1140. This operation can be performed such that when therotor chamber 1600 is filled with cells/media mixture, the pump 1140will stop. For example, with a peristaltic pump, if the rotor of theperistaltic pump stops rotating, there will be no further flow and therewill be no backflow. So, the pump 1140 can simply be shut off and theliquid can be allowed to remain where it is at that time, including theliquid level in the culture region. It is possible that the volumes ofliquid may have been calculated and measured sufficiently accurately sothat at that point the reservoir 1060 is nearly empty. In fact, it ispossible that the liquid level in the reservoir 1060 may have dropped tosomewhere in the reservoir sump recess 1440. If the liquid level is inthe reservoir sump recess 1440, lower than the upper surface 40 ofreservoir base 1400 but higher than the bottom end of riser conduit1500, the liquid inside the riser conduit 1500 and the cell cultureregion will be maintained in a stable position because there will be nopassage of air bubbles into the liquid column to allow liquid to fall.The cell/media will be maintained inside the riser column 1500 and thecell culture region by the reduced gas pressure (which may be slightlysub-atmospheric) above the cell culture region, even though the pump1140 is stopped at that time. A pump such as a peristaltic pump, orgenerally a positive-displacement pump, may maintain such a situation.Valving could also be used.

In the process of seeding, media containing cells may be raised bysuction into the region of the rotor 1700 and the cell culturescaffolds. For initial inflow of liquid, the position of the screens1300 may be generally vertical. The seeding process can then start byrotating the rotor 1700 so that the screens 1300 occupy a horizontalposition. This allows cells that are suspended in the liquid between thescreens 1300 to settle downward by gravity onto the adjacent screens1300. The scaffold (screen) 1300 may be maintained in a stationarycondition for a period of time as this happens. It is also possible thatat desired times, the rotor 1700 can again be rotated. For example, therotor 1700 might be rotated 180 degrees so that cells that might bemerely resting unattached on top of one screen 1300 might float away andsettle on and attach to a nearby screen 1300 due to the change of thegravitational direction. The rotor 1700 can be rotated periodically toproduce a uniform cell distribution on the screens or scaffold. Thisprocess may continue for as long as one to two days (to be determined byexperiment).

When seeding is completed, it is expected that all (or most) of thecells will be attached to the scaffolds (screens) 1300. The follow-upsteps may involve a more continuous flow of culture medium and can betermed dynamic culturing. Additional media can be added to the reservoir1060. This additional medium does not need to contain cells. The pump1140 may be turned on to allow dynamic culture by means of mediacirculation. The flow can flow continuously around the flow systemthrough the scaffold, if desired. The flowrate can be adjusted to be lowenough to keep the shear rate at the cell locations to a desirably smallmagnitude. At this point, there may be some cells that have not attachedto the scaffolds and that flow through the pump tubing. It is hoped toachieve at least 80% seeding efficiency in the seeding process.

The procedure as far as operation of the motor and positioning of thescaffold screens could be as follows: [0163] Fill the reservoir 1060with liquid that contains cells to be cultured and expanded. [0164] Forinitial filling of the culture region with liquid, position the screensvertically. [0165] After the initial filling of the culture region withliquid, position the screens (scaffold) 1300 horizontally so that cellsthat are in suspension between the screens can settle out of suspensiononto the screens. [0166] At intervals, rotate the screens 180 degrees sothat upward-facing screen surfaces become downward-facing screensurfaces and vice versa. The intervals might, for example, be one hour.[0167] Continue doing this for a sufficient time for a large fraction ofthe cells to have attached to the screens 1300. It is anticipated thatthis step might take about one to two days. [0168] After the cells arewell seeded, rotate the screens (scaffold) 1300 to the vertical positionor to the horizontal position as desired for dynamic culturing. [0169]During dynamic culturing, leave the screens (scaffold) 1300 in eitherthe vertical position or the horizontal position as desired. Placenutrients in the media, and slowly flow the media past the screens(scaffold) 1300 for the duration of dynamic culturing. The flow will beupward and generally parallel to or through (perpendicular to) the flatsurfaces of the screens 1300 as desired. [0170] After a sufficient time,harvest the cells.

In general, it is possible to execute any desired combination ofpositions of the rotor and scaffolds at particular stages of the seedingand cell culturing process. During dynamic culturing, the screens couldbe in either vertical or horizontal position during culturing, whicheveris desired, and the flow could be either parallel to or through(perpendicular to) the screens.

Harvesting of Cells

In embodiments of the invention, there may be practiced certaintechniques for harvesting cells from a scaffold 1300 after the cellshave grown. It is known that tryp-LE E is useful for harvesting cellsfrom a scaffold. In embodiments of the invention, for harvesting, ofcells, the scaffolds will be washed with PBS three times and followedwith addition of appropriate amount of tryp-LE E for about 30 minutes.Harvesting of cells may further be aided by the action of an impactorthat impacts the cytoskeleton of the cells that is structurallyconnected to the screens (or scaffold). This impacting can dislodgecells through the mechanical force of the impact.

Computational Fluid Dynamics

Referring now to FIGS. 19A-20 , there are shown results of ComputationalFluid Dynamics modeling of flow geometries of an embodiment of theinvention. FIG. 19A shows local velocity distributions at a flowrate of30 milliliters/minute (with a cross-sectional area in the incomingchannel of approximately 6200 mm 2). FIG. 19B shows a velocitydistribution for a flowrate of 40 milliliters/minute. FIG. 19C shows avelocity distribution for a flowrate of 60 milliliters/minute. FIG. 20shows pressure distribution and streamlines of flow between screens thatare constructed as described herein.

In general, any combination of disclosed features, components andmethods described herein is possible. Steps of a method can be performedin any order that is physically possible.

All cited references are incorporated by reference herein.

Although embodiments have been disclosed, it is not desired to belimited thereby. Rather, the scope should be determined only by theappended claims.

What is claimed is:
 1. A bioreactor system, comprising: a reservoircontainer for holding a liquid medium; a manifold assembly, saidmanifold assembly comprising an upper manifold and a lower manifold,said lower manifold having a lower end extending into said reservoircontainer; a holder, said holder being contained within said manifoldassembly, and said holder holding a plurality of fiber assemblies thatare suitable for cells to grow on; and a circulation system for causingthe liquid medium to flow through said lower manifold and said fiberassemblies and said upper manifold, wherein the liquid medium flowsthrough a sequence that comprises (a) one of said fiber assemblies,wherein said one of said fiber assemblies comprises a plurality of solidfibers that are joined to others of said solid fibers at crossing pointswith others of said solid fibers, followed by (b) an open region inwhich the liquid medium flows generally perpendicular to said one ofsaid fiber assemblies through space not occupied by any solid object,wherein said sequence repeats itself a plurality of times along adirection of flow of the liquid medium, wherein said fiber assembliesare held in said holder such that said fiber assemblies do not touch anyneighboring one of said fiber assemblies under static conditions and donot touch any neighboring one of said fiber assemblies under conditionsof flow of the liquid medium during cell culture.
 2. The bioreactorsystem of claim 1, wherein said fiber assemblies have a fiber assemblythickness and said fiber assemblies are separated from a neighboring oneof said fiber assemblies by a separation distance, a direction of saidfiber assembly thickness and a direction of said separation distancebeing substantially aligned with each other, and wherein said separationdistance is between 1.5 and 2.5 times said fiber assembly thickness. 3.The bioreactor system of claim 1, wherein at least some of said fiberassemblies comprise a plurality of said fibers forming a first layerhaving said fibers parallel to each other and oriented in a firstdirection and a second layer having a plurality of said fibers parallelto each other and oriented in a second direction that is perpendicularto said first direction, said fibers in said second layer being joinedto said fibers in said first layer.
 4. The bioreactor system of claim 1,wherein at least some of said fiber assemblies comprise a plurality ofsaid fibers, and within an individual one of said fiber assemblies, saidfibers are arranged in sequence in at least first, second, third andfourth layers, said fibers within each of said layers being generallyparallel to each other, wherein said fibers in said first layer aregenerally parallel to said fibers in said third layer and said fibers insaid second layer are generally parallel to said fibers in said fourthlayer, and wherein when viewed perpendicular to a flat surface of saidfiber assembly, said fibers of said third layer are located non-alignedwith said fibers of said first layer and said fibers of said fourthlayer are located non-aligned with said fibers of said second layer. 5.The bioreactor system of claim 1, wherein said fiber assemblies have abending stiffness equal to or greater than a bending stiffness such thatif one of said fiber assemblies, having a width of 60 mm, is simplysupported at two points 100 mm apart and is loaded by 14.5 grams, itsdeflection at center span is 3.1 mm.
 6. The bioreactor system of claim1, wherein said upper manifold and said lower manifold and said holderhave respective internal cross-sectional flow areas that aresubstantially identical to each other or are within 10% of each other.7. The bioreactor system of claim 1, wherein a leakage path comprising agap between an individual one of said fiber assemblies and said holder,has a cross-sectional flow area that is less than 10% of a total flowarea through all passageways through said individual one of said fiberassemblies.
 8. The bioreactor system of claim 1, wherein said lower endof said lower manifold has a first perimeter and said lower manifoldlower end is at a uniform elevation around an entirety of said firstperimeter, and wherein an upper end of said upper manifold has a secondperimeter and said upper manifold upper end is at a uniform elevationaround an entirety of said upper manifold upper end second perimeter. 9.The bioreactor system of claim 1, wherein said reservoir containercontains therewithin a contoured filler piece at least some of which islower than said lower end of said lower manifold, said contoured fillerpiece being shaped so as to smoothly direct flow of the liquid mediumfrom said reservoir container into said lower manifold, said contouredfiller piece being wider at its bottom and narrower at its top.
 10. Abioreactor system, comprising: a reservoir container for holding aliquid medium; a manifold assembly, said manifold assembly comprising anupper manifold and a lower manifold, said lower manifold having a lowerend extending into said reservoir container; a holder, said holder beingcontained within said manifold assembly, said holder holding a pluralityof fiber assemblies that are suitable for cells to grow on; and acirculation system for causing the liquid medium to flow through saidlower manifold and said fiber assemblies and said upper manifold,wherein an internal cross-sectional dimension of said lower manifoldbelow said holder, an internal cross-sectional dimension of said uppermanifold above said holder, and an internal cross-sectional dimension ofsaid holder are all substantially equal to each other, and wherein aside-to-side dimension of said fiber assembly is larger than acorresponding inside dimension of said holder, and said side-to-sidedimension of said fiber assembly is smaller than a corresponding insidedimension of said manifold assembly in which said holder resides. 11.The bioreactor system of claim 10, wherein an inner wall of said lowermanifold and an inner wall of upper manifold and an inner wall of saidholder are substantially parallel to and aligned with each other. 12.The bioreactor system of claim 10, wherein said fiber assemblies areheld in said holder such that said fiber assemblies do not touchneighboring ones of said fiber assemblies under static conditions and donot touch neighboring ones of said fiber assemblies under conditions offlow of the liquid medium during cell culture.
 13. The bioreactor systemof claim 10, wherein said holder comprises two parts joinable to eachother by engagement features and wherein said engagement features do notprotrude interiorly beyond walls of said holder, and wherein one of saidengagement features has therethrough a groove that is continuous with agroove in an adjacent portion of an interior surface of said holder. 14.The bioreactor system of claim 10, wherein a portion of said uppermanifold slidingly engages with a portion of said lower manifold andwherein an upper surface of said holder faces a corresponding surface ofsaid upper manifold and a lower surface of said holder faces acorresponding surface of said lower manifold.
 15. The bioreactor systemof claim 10, wherein said reservoir container is covered by a reservoircover, and said reservoir cover has therein an opening having a raisededge or lip surrounding said opening, and said lower manifold hasthereon a flange suitable to contact said lip, and said flange extendsbeyond said lip such that possible condensate on an external surface ofsaid manifold assembly cannot drip into said reservoir container. 16.The bioreactor system of claim 10, wherein said fiber assemblies arerestrained within said holder but have at least some ability to movewith respect to said holder in at least one direction.
 17. Thebioreactor system of claim 16, wherein said fiber assemblies have atleast some ability to move with respect to said holder in three mutuallyorthogonal directions.
 18. The bioreactor system of claim 10, whereinwhen said holder is in place within said manifold assembly, said holderhas at least one degree of freedom of motion with respect to said lowermanifold.
 19. The bioreactor system of claim 10, wherein at least someof said fiber assemblies comprise a plurality of solid fibers forming afirst layer having said fibers parallel to each other and oriented in afirst direction and a second layer having a plurality of said fibersparallel to each other and oriented in a second direction that isperpendicular to said first direction, said fibers in said second layerbeing joined to said fibers in said first layer.
 20. A method ofculturing cells, said method comprising: providing a bioreactor, saidbioreactor comprising: a reservoir container for holding a liquidmedium; a manifold assembly, said manifold assembly comprising an uppermanifold and a lower manifold, said lower manifold having a lower endextending into said reservoir container; a holder, said holder beingcontained within said manifold assembly, said holder holding a pluralityof fiber assemblies that are suitable for cells to grow on; and acirculation system for causing the liquid medium to flow through saidlower manifold and said fiber assemblies and said upper manifold,wherein the liquid medium flows through a sequence that comprises (a)one of said fiber assemblies, wherein said one of said fiber assembliescomprises a plurality of solid fibers that are joined to others of saidsolid fibers at crossing points with others of said solid fibers,followed by (b) an open region in which the liquid medium flowsgenerally perpendicular to said one of said fiber assemblies throughspace not occupied by any solid object, wherein said sequence repeatsitself a plurality of times along a direction of flow of the liquidmedium, wherein said fiber assemblies are held in said holder such thatsaid fiber assemblies do not touch any neighboring one of said fiberassemblies under static conditions and do not touch any neighboring oneof said fiber assemblies under conditions of flow of the liquid mediumduring cell culture, wherein an internal cross-sectional dimension ofsaid lower manifold below said holder, an internal cross-sectionaldimension of said upper manifold above said holder, and an internalcross-sectional dimension of said holder are all substantially equal toeach other, and wherein a side-to-side dimension of said fiber assemblyis larger than a corresponding inside dimension of said holder, and saidside-to-side dimension of said fiber assembly is smaller than acorresponding inside dimension of said manifold assembly in which saidholder resides; seeding cells onto said fiber assemblies; operating saidcirculation system under conditions appropriate for the cells tomultiply into a plurality of cultured cells; and harvesting the culturedcells from said bioreactor.